Polyubiquitin based hydrogel and uses thereof

ABSTRACT

The present invention relates to a novel biopolymer consisting of a three-dimensional cross-linked mixture of (a) a cross-linking agent, activated with an activating agent, dissolved in a aqueous solution, and (b) a recombinant protein, namely polyubiquitin. The novel biopolymer is based on the cross-linking of ubiquitin (monomeric and/or polymeric) with a cross-linking agent, preferably bifunctionalized polyethylene oxides or a polyethylene glycol of various molecular masses (MW 2000 to 35 000 kDa), dissolved in aqueous solution in adequate proportions. The novel biopolymer offers a wide range of formulations since the number of ubiquitin units and cross-linking agent can vary both in length and ratio. The novel hydrogel is also biodegradable by a specific protease and is resistant to a wide range of other proteases.

BACKGROUND OF THE INVENTION

[0001] (a) Field of the Invention

[0002] The invention relates to a bioartificial hydrogel composed of apolymer of ubiquitin units cross-linked with a bifunctionnalpolyethylene glycol and derivatives thereof, such as polyethylene oxide,in an aqueous solution. The forming polyubiquitin hydrogel can be usedas a wound dressing as a biodegradable delivery vehicle for the systemicor topic delivery of bioactive agents. The hydrogel can also be usedalso as a biosensor of enzymes, for detection of different nucleic orpeptidic molecules. It is defined as a detection condition sensitivesystem. It further relates to an in situ hybridization system.

[0003] (b) Description of Prior Art

[0004] Techniques have been developed for administering pharmaceuticalsthrough the skin by absorption. Such techniques are accomplished bydevices which typically comprise either a pharmaceutical-containingreservoir enclosed by a synthetic membrane through which thepharmaceutical can diffuse at a controlled rate, or a dispersion of apharmaceutical in a synthetic polymer matrix in which the pharmaceuticalcan diffuse at a controlled rate. While such delivery devices work forsome pharmaceuticals, the rate of release of other pharmaceuticals isnot adequate through synthetic polymers. Either the rate of delivery istoo slow to provide an effective dosage given the area of the deliverysurface, or in some cases, where prolonged delivery of the drug isdesired, delivery is too fast so that the device must be replaced withina short period of time. One situation in which it is desirable to have adrug delivered over a prolonged period of time without removal of thedelivery device is the case of delivery of drugs at a wound site arounda percutaneous medical device.

[0005] Moreover, it is desirable, particularly when dealing withdelivery of bioactive agents that are natural products, such as growthfactors, that the polymeric matrix from which the drug is delivered betailored for optimal drug delivery rate. it is difficult to do this whenthe drug to be delivered is a biological macromolecule, such as anenzyme or surface receptor, since specialized binding functionalitieswith proper charge density, orientation, hydrophobic domains, etc. arenot readily synthesized into synthetic polymers to release thebiological macromolecule at a desired controlled rate.

[0006] U.S. Pat. No. 4,101,380, the specification of which isincorporated herein by reference, discloses a wide variety of reagentsuseful to activate polyethylene oxide in the object of obtaining abifunctionalyzed polyethylene oxide or polyalkene oxide. When thosereagents are used to cross-link PEG with a gelatin preformed membrane, across-linked gelatin-PEG membrane was obtained and was characterized bya high liquid swelling capacity. However, other embodiments described inthe patent provided very low yield of protein cross-linking (in theorder of about 2%). The patent states that the use of a carbonatederivative of polyethylene oxide is not recommended and not useful.Attempts should be made to obtain cross-linking of the polymer with aprotein or enzyme. This is explained as being due to the high pHrequired for the subsequent cross-linking reaction which could inducedenaturation of enzymes or proteins.

[0007] U.S. Pat. No. 5,733,563 discloses albumin based hydrogel formaking contact lenses, controlled drug release devices, immobilizationmatrix for enzymes or cells of therapeutic interest as enzymecorrection, wound dressing and artificial skin. The hydrogel containspolyethylene glycol cross-linked with albumin from various sources.Meanwhile, the hydrogel of this invention is characterized by the use ofalbumin, which gives the possibility to produce hydrogel having only onedensity. Other limitations of this hydrogel are low resistance totemperature and pH variations, high vulnerability to a great number ofproteolytic enzymes, and high potential of inducing allergic reactions.

[0008] U.S. Pat. No. 4,615,697 discloses the use of a polymer asmoisturizer and humectant and as a bioadhesive vehicle for thecontrolled release of active principles, in the pharmaceutical field.The synthetic polymer is Polycarbophil, a polyacrylic acid cross-linkedwith divinyl glycol (3,4-dihydroxy-1,5-hexadiene).

[0009] U.S. Pat. No. 5,891,558 features biopolymer foams, compositebiopolymer foams, biocompatible constructs comprising biopolymer foamsand extracellular matrix particulates and methods for making and usingthese foams and foam compositions. The foams and foam compositions canbe used in vitro, for example, for model systems for research, or invivo. In either case, the foam compositions can be seeded with cells,e.g., mammalian cells, e.g., human cells, of the same type as those ofthe tissue which the foams or foam compositions is used to repair orreconstruct. However, collagen sponges, gelatin sponges or polyvinylalcohol sponges lack biological activity typically present in theextracellular matrix environment of cells, and because of theirdeficiencies, cross-linked collagen sponges induce little regenerationin vivo or serve poorly as histiotypic and organotypic models in vitro.

[0010] U.S. Pat. No. 6,039,940 incorporated herein by referencediscloses composition and method for treating a wound with an inherentlyantimicrobial dressing. The dressing is an hydrogel containing fromabout 15 to 95 percent, and preferably from about 61 to 90 percent, byweight of a cationic quaternary amine acrylate polymer prepared by thepolymerization of acryloyloxyethyl (or propyl)-trialkyl (oraryl)-substituted ammonium salts or acrylamidoethyl (or propyl)-trialkyl(or aryl)-substituted ammonium salts. The antimicrobial hydrogels arenon-irritating to the wound, absorb wound exudate, and, due to theinherently antimicrobial properties, enhance the sterile environmentaround the wound.

[0011] Also, the application of recombinant DNA techniques is emergingas a powerful tool in the area of molecular diagnostic medicine. Forexample, the development of DNA and RNA molecular probes for thedetection of viral and bacterial genomes and genetic defects inmammalian chromosomes may replace current immunochemical approaches.

[0012] Polynucleotide hybridization assays are used as research toolsfor the detection and identification of unique or specificpolynucleotide sequences in samples of complete, fragmented, or mixednucleic acids. Various hybridization diagnostic techniques have beendeveloped.

[0013] The southern blot technique is based on a polynucleotidehybridization technique employing radiolabeled nucleic acid probes. Thisprocedure permits autoradiographic detection of probe/analyte hybridsand identification of the polynucleotide sequence of the analyte.However, the Southern procedure, as well as the other diagnosticprocedures employing radiolabeled nucleic acid probes, are very complex,time consuming, and have the additional problems and expenses generallyassociated with radioactive materials such as disposal and personnelmonitoring. Thus, such assays have remained a tool of basic research andare not generally employed in applied or commercial areas such asclinical diagnosis.

[0014] Most of the existing methods used to attach a polynucleotideprobe to a solid support are non-specific and the number of attachmentsites per nucleic acid is difficult to control. It has been found thatmultiple attachment reduces the degree of freedom of the immobilizednucleic acid. The physical adsorption of single stranded DNA, covalentattachment via diazo-linkage, epoxidation, cyanogen bromide activationand photochemical reactions are associated with the complication ofnon-specific linkage between the nucleic acids and the solid support.

[0015] Canadian Patent No. 1,223,222, which is incorporated herein byreference, discloses an immobilized nucleic acid-containing probecoupled to a solid support in a manner which is site specific, whichdoes not interfere with the ability of the nucleic acid to hybridize andwhich involves preferably a single chemical covalent linkage per nucleicacid to the solid support. Specifically, the nucleotide is coupled tothe nucleic acid employing an enzyme and the nucleotide is chemicallymodified.

[0016] Canadian Patent No. 1,293,937 discloses polynucleotide probecompositions, diagnostic kits, and nonradiometric hybridization assaysuseful in the detection and identification of at least one targetpolynucleotide analyte in a physiological sample. There is provided afirst polynucleotide probe having a catalyst attached thereto and whichis substantially complementary to a first single-stranded region of theanalyte and a second polynucleotide probe having an apoluminescerattached thereto and which is substantially complementary to a secondsingle-stranded region of the analyte. The second region issubstantially mutually exclusive from the first region, such that uponhybridization of the first and second probes with the analyte, thecatalyst and the apoluminescer are close enough to each other to permitthe catalyst to act on a substrate to release a transformation radicalto convert the apoluminescer to a luminescer.

[0017] Current methods for the diagnosis of inherited diseases employdigestion of a prepared DNA sample with restriction enzymes to formshort, double-stranded segments, gel electrophoresis to separate thesesegments according to size, transfer of the separated segments to a thinmembrane material, such as nylon, hybridization of the segments ofinterest with a labeled oligonucleotide (of complementary sequence tothe known disease sequence), and detection of the label. The completeprocedure requires about 24 hours, is labor-intensive, and is notreadily automated. Furthermore, these methods usually employ radioactivelabels, with their inherent safety and disposal problems. None of theabove-mentioned diagnostic systems discloses a probe that can be treatedto be reusable for hybridization. Thus, these systems are for a uniqueusage.

[0018] A significant drawback in the use of hydrogels, however, and onethat has substantially hindered the use of hydrogels in drug deliverysystems, is that such formulations are generally not biodegradable.Thus, drug delivery devices formulated with hydrogels typically have tobe removed after subcutaneous or intramuscular application or cannot beused at all if direct introduction into the blood stream is necessary.Thus, it would be advantageous to use hydrogels that could be degradedafter application in the body without causing toxic or other adversereactions.

[0019] In the art mentioned above, there is no mention or suggest thatadvantageous hydrogels could be obtained by cross-linking of apolyubiquitin or another native protein in an aqueous solution withactivated polyethylene oxide. Therefore, it would be highly desirable tobe provided with an improved hydrogel that overcomes or minimizes theabove-mentioned problems.

[0020] It would also be highly desirable to be provided with abiodegradable hydrogel that has significantly enhanced biocompatibilityin that (1) blood compatibility is substantially improved, (2)immunogenicity is minimized, and (3) the hydrogel is enzymaticallydegraded to endogenous, nontoxic compounds.

SUMMARY OF THE INVENTION

[0021] One object of the present invention is to provide biopolymercomprising a mixture of ubiquitin and cross-linking agents.

[0022] Another object of the present invention is to provide biopolymerwherein the cross-linking agent may be photoreactive or thermoreactive.A thermoreactive cross-linking agent is a compound that may contain athermochemical reactive group that may be a —COOH (carboxylic acids),sulfonic acid derivatives, —COOR (esters), —COX (acid halides, acidazides and similar carboxylic acid derivatives), —CONHNH₂ (acidhydrazides), —NHCONHNH₂ (semicarbazides), —NHCSNHNH₂(thiosemicarbazides), —CHO (aldehydes), RR′CO (ketones), —OH (alcohols),—X (halides: chloride, bromide, iodide), —SH thiols, —SSR (disulfides),—NH₂ (primary amines), —NH— (secondary amines), —N— (tertiary amines),—NHNH₂ (hydrazines), epoxides, and maleimides.

[0023] A further object of the present invention is to provide with abiopolymer having ubiquitin that may be found under forms of ubiquitinunit, or tandem of ubiquitin units comprising between 2 to about 25ubiquitin units and combination thereof. The ubiquitin may be purifiedfrom natural sources, recombinant, mutant, analog, fragment, andderivative thereof.

[0024] The cross-linking agent of the invention may comprise apolyethylene glycol, or other cross-linking agent which may consist ofpolyamine, amine, polyvinyl, polystyrene, epoxy, silicone,proteinaceaous, keratin, collagen, elastin, actin, myosin, fibrinogen,silk, polysaccharides, cellulose, amylose, hyaluronic acid, gelatin,chitin, chitosan, xylan, mannan, silica, and derivative thereof.

[0025] Another object of the invention is to provide a cross-linkingagent that is a derivative of polyethylene glycol, namely polyethyleneoxide derivatives, or bifunctionalized polyethylene oxide, of thegeneral formula 1:

—X—(CH₂—CH₂—O)_(n)—X

[0026] wherein n is at least 1; X is a covalent bound or capable ofreacting with an amino acid, or is an R or RO radical in which theoxygen is bound to the polyethylene oxide and R is selected from thegroup of methylene, ethylene, propylene, o-, m- and p-phenylene, o-, m-and p-phenylene carbamate unsubstituted or substituted by at least onealkyl, aryl, halo, nitro, oxo, carboxy, hydroxy, thio, sulfonate,hydroxy and phosphate groups.

[0027] Another object of the invention is to provide a process forpreparing a ubiquitin biopolymer, by mixing a ubiquitin solution with atleast one cross-linking agent, and inducing polymerization between theubiquitin in solution and the cross-linking agent for a time sufficientfor a cross-linking reaction to occur.

[0028] The ubiquitin used in the process may comprise ubiquitin units,or tandem of ubiquitin units that may contain between 2 to 25 ubiquitinunits and combination thereof.

[0029] The process for making the novel hydrogel represents a furtheradvance over the art in that, during synthesis, one can carefullycontrol factors such as hydrophilicity, charge and degree ofcross-linking. By varying the composition of the hydrogel as it is made,one can control the uptake of a particular drug, the degradationkinetics of the hydrogel formulation and the overall timed-releaseprofile.

[0030] Also, the cross-linking agent used for the process of the presentinvention may be photoreactive, or thermoreactive cross-linking agent,wherein thermoreactive compound is a compound containing athermochemical reactive group that may be selected from the groupconsisting of: —COOH (carboxylic acids), sulfonic acid derivatives,—COOR (esters), —COX (acid halides, acid azides and similar carboxylicacid derivatives), —CONHNH₂ (acid hydrazides), —NHCONHNH₂(semicarbazides), —NHCSNHNH₂ (thiosemicarbazides), —CHO (aldehydes),RR′CO (ketones), —OH (alcohols), —X (halides: chloride, bromide,iodide), —SH thiols, —SSR (disulfides), —NH₂ (primary amines), —NH—(secondary amines), —N— (tertiary amines), —NHNH₂ (hydrazines),epoxides, and maleimides.

[0031] The process of the present invention may comprise ubiquitinpurified from natural sources, or may be recombinant, mutant, analog,fragment, and derivative thereof.

[0032] The present process may comprise as cross-linking agentpolyethylene glycol, or a derivative of polyethylene glycol, such aspolyethylene oxide, or an activated bifunctionalized polyethylene oxideof the general formula 1:

X—(CH₂—CH₂—O)_(n)—X

[0033] wherein n is at least 1; X is a covalent bound or capable ofreacting with an amino acid, or is an R or RO radical in which theoxygen is bound to the polyethylene oxide and R is selected from thegroup of methylene, ethylene, propylene, o-, m- and p-phenylene, o-, m-and p-phenylene carbamate unsubstituted or substituted with at least onealkyl, aryl, halo, nitro, oxo, carboxy, hydroxy, thio, sulfonate,hydroxy and phosphate groups.

[0034] The process may also comprise cross-linking agent selected fromthe group consisting of polyamine, amine, polyvinyl, polystyrene, epoxy,silicone, proteinaceaous, keratin, collagen, elastin, actin, myosin,fibrinogen, silk, polysaccharides, cellulose, amylose, hysluronic acid,gelatin, chitin, chitosan, xylan, mannan, silica, and derivativethereof.

[0035] Another object of the invention is to provide a biopolymerconsisting essentially of ubiquitin, which may comprise ubiquitin unit,or tandem of ubiquitin units comprising between 2 to about 25 ubiquitinunits and combination thereof. Combinations used to compose biopolymersmay mean, for example but not limited to, combinations of tandems of nubiquitin units with tandems of x ubiquitin units, wherein n and xrepresents between 2 to 25. There may be a combination of tandemscomposed of 7 ubiquitin units with tandems composed of 15 ubiquitinunits, for example.

[0036] Another object of the present invention is the use of ubiquitinin the preparation of a biopolymer.

[0037] For the purpose of the present invention the following terms aredefined below.

[0038] The term “biologically active” is intended to mean a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule.

[0039] The term “polypeptide” is intended to mean a given amino acidsequence, as these terms are used herein, refer broadly to the presenthydrogel containing the given polynucleotide or amino acid sequence. Thehydrogel may comprise a dry formulation or an aqueous solution. Hydrogelcomprising polynucleotide sequences may be employed as hybridizationprobes.

[0040] The term “polyubiquitin” as used herein means tandem repeats ofubiquitin unit, with the number of repeats varying from 2 to 20, andvarying naturally also between species. The DNA encoding sequence ofpolyubiquitin is the ubiquitin fusion gene, which encodes ubiquitinunits in head-to-tail array arrangements.

[0041] The term “targeted molecule” or “targeted marker” is intended tomean a molecule to be detected or dose in a biological sample. Thisinvolved, without limitation, DNA or RNA sequences, proteins,polypeptides, and any other amino acid sequence of any length.

[0042] The term “biological sample” as used herein means a biologicalfluid, tissue, or mater containing cells, proteins, DNA or RNAsequences, polypeptide, proteins, oligopeptides, and any other aminoacid sequence of any length. The fluid may include, but is not limitedto, tears, saliva, milk, urine, amniotic fluid, semen, plasma, serum,oviductal fluid, and synovial fluid. The tissues may include, but arenot limited to, lung, heart, blood, liver, muscle, brain, pancreas,skin, and others. The biological sample may origin from an animal, aplant, bacteria, yeast, or any living organism. The biological samplemay become, but is not limited to, an in vitro culture of eucaryote orprocaryote cells, or any other amplification procedures.

[0043] The term “hybridization” as used herein, refers to any process bywhich a strand of nucleotidic acid, or polynucleotide, binds with acomplementary strand through base pairing, or biochemical affinity.

[0044] The terms “nucleic acid” or “nucleic acid sequence” as usedherein, refer to an oligonucleotide, nucleotide, polynucleotide, or anyfragment thereof, to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded an may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In the context, “fragments” refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

[0045] The term “oligonucleotide” as used herein, refers to a nucleicacid sequence of at least about 6 nucleotides to 60 nucleotides,preferably about 15 to 30 nucleotides, and most preferably about 20 to25 nucleotides, which can be used in PCR amplification or in ahybridization assay or microssay. As used herein, the term“oligonucleotide” is substantially equivalent to terms “amplimers”,“primers”, “oligomers”, and “probes”, as these terms are commonlydefined in the art.

[0046] The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

[0047] The terms “complementary” or “complementarity,” as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

[0048] The term “microarray,” as used herein, refers to an array ofdistinct polynucleotides or oligonucleotides arrayed on a substrate,such as paper, nylon or any other type of membrane, filter, chip, glassslide, or any other suitable solid support.

[0049] “Peptide nucleic acid” (PNA), as used herein, refers to anantisense molecule or antigen agent which comprises an oligonucleotideof at least about 5 nucleotides in length linked to a peptide backboneof amino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell.

[0050] The term “sample,” as used herein, is used in its broadest sense.A biological sample suspected of containing a molecule to be detected ordosed, nucleic acids or polypeptides, proteins, or fragments thereof,that may be comprised in a bodily fluid; tissue, an extract from a cell,chromosome, organelle, or membrane isolated from a cell; a cell; genomicDNA, RNA, or cDNA (in solution or bound to a solid support); a tissue; atissue print; an in vitro culture medium, and the like.

[0051] The term “cytokine” includes but is not limited to growthfactors, interleukins, interferons and colony stimulating factors. Thesefactors are present in normal tissue at different stages of tissuedevelopment, marked by cell division, morphogenesis and differentiation.Among these factors are stimulatory molecules that provide the signalsneeded for in vivo tissue repair. These cytokines can stimulateconversion of an implant into a functional substitute for the tissuebeing replaced. This conversion can occur by mobilizing tissue cellsfrom similar contiguous tissues, e.g., from the circulation and fromstem cell reservoirs. Cells can attach to the prostheses, which arebioabsorbable and can remodel them into replacement tissues.

[0052] As used herein, the terms “specific binding” or “specificallybinding” refer to that interaction between a protein or peptide and anagonist, an antibody, or an antagonist. The interaction is dependentupon the presence of a particular structure of the protein recognized bythe binding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

[0053] The term “matrix” as used herein is intended to mean capsule,tablets, films, microspheres, hydrogel, or the like. The matrix formedby a mixture of ubiquitin and cross-linking agents may serve as drugreservoir, drug delivery system, biosensor, and skin and wound sealer.The compositions formulated using the matrices can include conventionalpharmaceutical carriers or excipients, adjuvants, etc. Matrices in theform of discs, slabs or cylinders can be used as implants, whilemicrospheres can be applied as subcutaneous, intramuscular, intravenousor intra-arterial injections.

[0054] By “hydrogel” as used herein is meant a water-swellable,three-dimensional network of macromolecules held together by covalentcross-links. (These covalent cross-links are sometimes referred toherein as providing a “network linkage”, within the macromolecularstructure.) Upon placement in an aqueous environment, these networksswell to the extent allowed by the degree of cross-linking.

[0055] By the term “pharmacologically active agent” or “drug” as usedherein is meant any chemical material or compound suitable foradministration which induces a desired systemic or local effect. Ingeneral, this includes therapeutic agents in all of the majortherapeutic areas.

[0056] By “effective” amount of a pharmacologically active agent or drugis meant a non-toxic but sufficient amount of a compound to provide thedesired systemic or local effect.

[0057] The term “biopolymer” as used herein may be a polymer suitablefor introduction into a living organism, e.g., a human. The biopolymeris usually non-toxic and bioabsorbable when introduced into the livingorganism, and any degradation products of the biopolymer might be alsonon-toxic to the organism. The biopolymer can be formed intobiocompatible constructs that include, for example, biopolymer hydrogel,e.g., variable density matrix, and/or biopolymer particles.

[0058] Biopolymers, such as hydrogel or matrices are very useful invitro to provide model systems for research, or in vivo as hemostaticagents, scaffolds or as prostheses and implants to replace damaged ordiseased tissues. In both in vivo and in vitro applications, the matrixmay be seeded with various cell types, allowing in vitro study of cellfunctions in three dimensions, and promoting in vivo remodeling andintegration of the implant or prosthesis. Often a biopolymer constructthat includes a biopolymer matrix is prepared in vitro, such as byseeding the matrix with cells and culturing the growth anddifferentiation of these cells, prior to use in vivo.

[0059] The immobilized biopolymers may be subsequently exposed to one ormore chemical probes, i.e., probes are hybridized to targeted sequencesin the adsorbed biopolymers, if present. Until recently, hybridizationagents contained radioisotopes. Specific biomolecules or biomolecularsequences were detected visually by radiometric development of images onphotographic films placed in contact with the media containing theimmobilized, derived biomolecules. Radioimmunoassay methods have nowbeen supplemented with new, nonradiometric approaches includingchemiluminescent, fluorescent and calorimeter methods of detection, orwith polymerase chain reaction (PCR) methods of greatly amplifyingspecific nucleic acid sequences, or with combinations of thesetechniques. The chemiluminescent, fluorescent and colorimetric methodsof detection have not profoundly displaced radioimmunoassay methods,despite environmental and regulatory concerns about the handling ofradioactive chemicals. A drawback limiting the full-scale adoption ofthese newer methods is been their generally lower level of sensitivityversus radioimmunoassay sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060]FIG. 1 illustrates a slide cover for in situ hybridization (ISH),in situ PCR or immunohistochemistry (IHC);

[0061]FIG. 2 illustrates a piece of polyubiquitin hydrogel (PUH);

[0062]FIG. 3 shows according to one embodiment of the present invention,the molecular network relation between units of ubiquitin in theformation of a gel;

[0063]FIG. 4 shows an electron microscope view of PUH nanospheres;

[0064]FIG. 5 shows a second electron microscope view of PUH nanospheresat higher magnification;

[0065]FIG. 6 illustrates a Sensor 1 and a Sensor 2 that can be formed inpolymerizing units of ubiquitin with antibodies capable of catchingantigens;

[0066]FIG. 7 illustrates absorbency profiles of gels formed with PUH orBSA;

[0067]FIG. 8 illustrates transmittance profiles of gels formed with PUHor BSA;

[0068]FIG. 9 illustrates the UV absorbency of PUH at differenttemperatures;

[0069]FIG. 10 illustrates the optical absorbency of PUH at differenttemperatures and wavelengths;

[0070]FIG. 11 illustrates the optical absorbency variation of PUH inrelation with salt changes;

[0071]FIG. 12 illustrates the optical absorbency variation of PUH inrelation with salt changes and time;

[0072]FIG. 13 illustrates fluorescence emitting units of PUH and BSAgels in relation with pH changes;

[0073]FIG. 14 shows stained nanospheres observed under opticalmicroscope;

[0074]FIG. 15 illustrates according to one embodiment of the presentinvention, an enzymatic amplification using immobilized HRP comprisingthe relation between ligand molecule (a), immobilized enzyme (b) and PUH(c);

[0075]FIG. 16 illustrates effects of the dilution of PUH nanospheres onthe optical density of suspensions;

[0076]FIG. 17 illustrates the systemic release of insulin aftersubcutaneous administration of PUH containing insulin;

[0077]FIG. 18 illustrates PUH in humidifying chambers;

[0078]FIG. 19 illustrates epithelial cells stained with hematoxylin; and

[0079]FIG. 20 illustrates the release of dexamethasone from hydrated ordehydrated PUH.

DETAILED DESCRIPTION OF THE INVENTION

[0080] In accordance with the present invention, there is provided a newpolyubiquitin hydrogel (PUH) that can be used for several applications,most particularly as a drug delivery system, an enzymatic reactormatrix, a DNA, RNA, or antibody hybridization matrix, or as biosensor.

[0081] The ubiquitin, a small protein consisting of 76 amino acids, hasbeen found in all eukaryotic cells studies, it is one of the mostconserved proteins known; the amino acid sequence is identical frominsects to humans, and there are only 3 substitutions within the plantand yeast sequences. Two classes of ubiquitin genes are recognized.Class 1 is a polyubiquitin gene encoding a polyprotein of tandemlyrepeated ubiquitins. The class 2 genes are fusion products between asingle ubiquitin gene and 1 of 2 other possible sequences, either 52 or76 or 80 predominantly basic amino acids. Ubiquitin is required forATP-dependent, non-lysosomal intracellular protein degradation, whicheliminates most intracellular defective problems as well as normalproteins with a rapid turnover. Degradation involves covalent binding ofubiquitin to the protein to be degraded, through isopeptide bonds fromthe C-terminal glycine residue to the epsilon-amino groups of lysyl sidechains. Presumably, the function of ubiquitin is to label the proteinfor disposal by intracellular proteases.

[0082] The structure of ubiquitin is 3 to 5 turns of a α-helix atresidues 23 to 34, a short 3₁₀-helix at 56 to 59 and a mixed β-sheetwith five strands. Two of those strands are parallel and in the insideof the molecule at positions 1 to 7 and 64 to 72. The rest three strands10 to 17,40 to 45, and 48 to 50 are antiparallel. The β-strands areleft-handed and the α-helix fits in the cavity formed by the sheets.Also in the structure there are two GI, β-bulges. The first is betweenantiparallel β-strands and is made by Gly10, Lys11, and Thr7. The secondbulge is at two parallel strands (64 to 720) and is made by Glu64,Ser65, and Glu2. This bulge is very rare. In the molecule there are alsoseven reverse turns. The longest of those hydrogen bond (4 to 10) isThr7-Gly10. Also in Phe45-Ser65 there are four reverse turns and a small3₁₀-helix.

[0083] According to one embodiment of the present invention is theinteraction of ubiquitin polymers, polyubiquitin, with the water solubleform of the polyethylene glycol, namely the polyethylene oxide (PEO), orbifunctionalized polyethylene oxide, and derivatives thereof. The PEOacts as cross-linking agent by having on both extremities covalentbonds, or R or RO radical in which the oxygen is bound to thepolyethylene oxide —(CH₂—CH₂O)— and R is one ubiquitin unit or a polymerof 2 to 50 ubiquitin units. The polyethylene glycol is activated to formthe bifunctionalized polyethylene oxide derivatives having the generalformula Y—O—(CH₂—CH₂O)_(n)—Y, where Y can be any type of functionalizedgroups able to react with an amino, a S—H, an OH or a COOH group broughtby a protein, and n can vary from 45 to 800 which corresponds tocommercial Polyethylene glycol for which the molecular weight can varyfrom 2,000 to 35,000.

[0084] Another point of interest is the COOH terminal of the unit. Whenubiquitin is partially digested it gives ubiquitin-74 and glycylglycine.The complete amino acid sequence of a ubiquitin unit is: (SEQ ID NO:1)      1               5                 10NH2-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys             15                 20Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr-Ile-    30              35                  40Glu-Asn-Val-Lys-Ala-Lys-Ile-Gln-Asp-Lys-Glu-Gly-            45                   50Ile-Pro-Pro-Asp-Gln-Gln-Arg-Leu-Ile-Phe-Ala-Gly-     55                 55                  60Lys-Gln-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-                    65                  70Asn-Ile-Gln-Lys-Glu-Ser-Thr-Leu-His-Leu-Val-Leu-             75Arg-Leu-Arg-Gly-Gly-COOH

[0085] In one embodiment of the present invention there is provided aprocess for cross-linking of proteins. More specifically, the presentinvention relates to the novel use of new and known compounds forcross-linking of ubiquitin units or polyubiquitin polymers. In effectthe biopolymer of the present invention involves the use ofcross-linking agents falling into categories based on polyethylene oxidederivatives which compounds are in themselves known, and the ubiquitinunit or polymers thereof wherein the component are in themselves knownbut which heretofore have not been combined to form a hydrogel whenbound to cross-linking agents. Other cross-linking agents involvingpolyamine and polyamine derivatives, polysaccarides and derivativesthereof may be used in the formation of specific biopolymers orpolyubiquitin matrix. More precisely, products such as polyasdhehydes,N—O-dimthacryloylhydroxyamine, methylene diacrylate, divinyl glycol,cellulose and hydroxycellulose, collagen and collagen derivatives,chitosan, gelatin are all candidates in forming a polyubiquitinbiopolymer.

[0086] Among cross-linking agents of the present invention may be usedthermochemical-activable and photochemical-activable compounds.Thermochemical reactive groups are well-known in the art and are definedas functional groups, which are able to form covalent bonds tobiopolymer surfaces or ligands under conditions in which thephotochemically reactive group is non-reactive.

[0087] The thermochemical reactive groups may be —COOH (carboxylicacids), sulfonic acid derivatives, —COOR (esters, comprising activeesters), —COX (acid halides, acid azides and similar carboxylic acidderivatives), —CONHNH₂ (acid hydrazides), —NHCONHNH₂ (semicarbazides),—NHCSNHNH₂ (thiosemicarbazides), —CHO (aldehydes), RR′CO (ketones), —OH(alcohols), —X (halides: chloride, bromide, iodide), —SH (thioles), —SSR(disulfides), —NH₂ (amines, comprising primary, secondary and tertiaryamines), —NHNH₂ (hydrazines), epoxides, maleimides.

[0088] A number of photochemical methods of modifying polymer surfacesmay be used. In these methods a desired ligand, often a sensitivebiomolecule is immobilized on the biopolymeric material surface througha photochemically reactive group and a spacer.

[0089] In general, the covalent attachment of the desired molecule tothe surface can be established in three ways: 1) the photochemicallyreactive group, which is coupled, via a spacer to a thermochemicalreactive group is bound covalently to the surface by a photochemicalreaction. Subsequently, the desired molecule is coupled to the surfaceby thermochemical reaction. 2) The photochemically reactive group, whichis coupled, directly to the desired molecule is bound to the surface bya photochemical reaction. 3) The photochemically reactive group iscoupled covalently to the surface by a thermochemical reaction.Subsequently, the desired molecule is coupled to the surface by aphotochemical reaction. The same principle of coupling a cross-linkingagent and ubiquitin is exploited herein.

[0090] The first two strategies are potentially the most flexible onesand allow control of the orientation of the immobilized ligand. Asexample, when irradiated with UV light having a short wavelength, asecondary amine placed in the end position and coupled to psoralen canbe photochemically bound to a polystyrene surface. When biotin iscoupled to the spacer derivative, biotin can also be photochemicallybound to polymer surfaces or particles.

[0091] The disclosed latent reactive groups responsive to ultra-violet,visible or infrared portions of the electromagnetic spectrum are:azides, acylazides, azido formates, sulfonyl azides, phosphoryl azides;diazo compounds such as diazoalkanes, diazoketones, diazoacetates,beta-ketone-alpha-diazoacetates; aliphatic azo compounds, diazirines,ketone, diphenylketone and photoactivable ketones such as agent andubiquitin may be adjusted to optimize an application.

[0092] In another embodiment of the present invention, the hydrogelformulations contain a significant amount of polyethylene oxidecross-linked with ubiquitin units or ubiquitin polymers, generallyidentified as polyubiquitin.

[0093] According to another embodiment of the present invention, thereis provided a biopolymeric delivery compositions for controlled releaseof bioactive agents, particularly biological macromolecules, which isformed of a biopolymer and a synthetic polymer.

[0094] This invention relates to pharmaceutical compositions ofpharmacologically active polypeptides, or their encoding genes and cDNA,which provide continuous release of the polypeptide over an extendedperiod when the composition is placed in an aqueous, physiological-typeenvironment. The encoding nucleic acid sequences, DNA and RNA, could bereleased directly into a tissue or an organ from the polyubiquitinmatrix.

[0095] It has long been appreciated that the continuous release ofcertain drugs over an extended period following a single administrationcould have significant practical advantages in clinical practice, andcompositions have already been developed to provide extended release ofa number of clinically useful drugs, after oral dosing, parenteral, andtopical administration. A suitable method of parenteral administrationis the subdermal injection or implantation of a solid body, for examplea pellet or a film, containing the drug, and a variety of suchimplantable devices have been described. In particular, it is knownthat, for many drugs, suitable implantable devices for providingextended drug release may be obtained by encapsulating the drug in abiodegradable polymer, or by dispersing the drug in a matrix of such apolymer, so that the drug is released as the degradation of the polymermatrix proceeds.

[0096] In another embodiment of the present invention, there is providedan implantable or injectable pharmaceutical or veterinary formulationfor pharmacologically useful polypeptides, which is in solid form, andwhich absorbs water from the animal body, after implantation, to form ahydrogel from which the polypeptide is released continuously over anextended period of time.

[0097] Thus, according to the present invention, there is provided apharmaceutical delivery PUH composition comprising a pharmacologicallyuseful polypeptide and a pharmaceutically or veterinarily acceptableamphipathic, cross-linked, branch polymer, in which the component may bebiodegradable or hydrolytically unstable under normal physiologicalconditions, the composition being capable of absorbing water when placedin water or an aqueous physiological type environment.

[0098] This invention is applicable to polypeptides quite generally,without any limitation as to structure or molecular weight, but is mostuseful for polypeptides which are relatively hydrophilic, and thefollowing list, which is not intended to be exhaustive, is indicative ofpolypeptides which may be employed in the formulation of this invention:oxytocin, vasopressin, adrenocorticotrophic hormone (ACTH), epidermalgrowth factor (EGF), prolactin, luliberin or luteinizing hormonereleasing hormone (LH-RH), growth hormone, growth hormone releasingfactor, insulin, somatostatin, glucagon, interferon, gastrin,tetragastrin, pentagastrin, urogastrone, secretin, calcitonin,enkephalins, endorphins, angiotensins, renin, bradykinin, bacitracins,polymyxins, colistins, tyrocidin, gramicidines, and synthetic analoguesand modifications and pharmaceutically-active fragments thereof,monoclonal antibodies and soluble vaccines.

[0099] In one embodiment of the present invention, the PUH formingmatrix may include transforming growth factor-beta-1, platelet-derivedgrowth factor, basic fibroblast growth factor, syndecan-1, decorin,fibronectin, collagens, laminin, tenascin, and dermatan sulfate,syndecan-1, fibronectin, laminin, and tenascin. The matrix can alsoinclude cytokines, e.g., growth factors necessary for tissuedevelopment.

[0100] One embodiment of the invention is to provide a PUH matrix, orbiopolymer, which may play an instructive role, guiding the activity ofcells which are surrounded by it or which are organized on it. Since theexecution of cell programs for cell division, morphogenesis,differentiation, tissue building and regeneration depend upon signalsemanating from the matrix, three-dimensional scaffolds, such as PUH, areenriched with biologically active products, which exhibit the moleculardiversity and the microarchitecture of a generic extracellular matrix,and of extracellular matrices from specific tissues.

[0101] In another embodiment of the present invention, there is provideddrug delivery devices, particularly for wound dressings, containing suchpolymeric delivery vehicles for controlled release of antimicrobialand/or wound-healing agents to aid in the wound healing process.

[0102] The PUH maintains the wound in a moist condition that not onlyfacilitates healing but also enhances the cosmetic appearance of thewound as it heals.

[0103] As previously noted, in order to maintain or promote sterilityand enhance healing, an external antibiotic or other disinfectant hasbeen added to prior art hydrogels and/or wound dressings. While suchexternal antibiotics may still be added if it is deemed necessary, theinherent antimicrobial properties of the present hydrogels may make theadditions of such external additives unnecessary. As will be seen, theantimicrobial properties of the hydrogels of this invention areeffective agents against a wide range of microbes.

[0104] Another advantage of the PUH is sterilization. Suppliers ofdressings generally place them in a sealed environment in a sterilecondition. Because hydrogels are absorptive to steam and othersterilization agents, such as ethylene oxide, they cannot be sterilizedby such means and the use of radiation is inimical to the stability ofmany prior art gels due to free radical degradation. The hydrogels ofthe present invention can be irradiated and sealed without adverseeffects to the stability, adhesivity or antimicrobial properties of thehydrogel. Due to the ability of the hydrogels to be sterilized byradiation, they do not have to be formed or packaged in a “clean room”or sterile environment.

[0105] When using the PUH as wound dressings, the PUH may also contain abuffer system to help prevent discoloration and/or hydrolysis of thehydrogels, and/or improve their shelf life. Other additives may also beadded to the hydrogels either before or after curing (i.e.pharmaceuticals, humectants, plasticizers, etc.). The appropriateness ofsuch additives is generally dependent upon which dressings are to beformulated and applied to a wound.

[0106] As mentioned above, the present hydrogels may include a buffersystem to help control the pH, help prevent discoloration, and/or helpprevent breakdown due to the extended presence of water (i.e. helpprevent hydrolysis). Buffers, if any, are preferably added to themixture prior to curing. Suitable buffers include, for example, but arenot limited to, sodium potassium tartarate, and/or sodium phosphatemonobasic, both of which are commercially readily available from, forexample, Aldrich. Chemical Co., IN. The use of a buffer system with thepresent hydrogel is preferred to provide the hydrogel with acommercially suitable shelf life (i.e. a shelf life of over one-year)without discoloration.

[0107] As is also mentioned above, other additives may be included inthe present hydrogels either before or after curing (i.e.pharmaceuticals such as antibiotics, disinfectants and the like,humectants, plasticizers, etc.). The appropriateness of such additivesis generally dependent upon the intended end use of the particularhydrogel as a wound dressing.

[0108] The thickness of the polymeric matrix may be varied as desired,depending upon the desired pharmaceutical dosage and duration ofdelivery. Ordinarily, a suitable matrix thickness will be in a range ofabout 0.1 to 1.0 centimeters.

[0109] It will be realized from the teachings herein that for allapplications, the degree of cross-linking, thickness and/or shape of thecross-linked biopolymer, and the degree of porosity (if any) are allparameters which may be controlled to attain a desired release profileof the bioactive agent from the cross-linked biopolymer.

[0110] The shape of the cross-linked biopolymer may be formed by moldingor casting before cross-linking or, after cross-linking, it may beformed into a desired shape by cutting. The cross-linked biopolymer willthen be loaded with the desired bioactive agent(s), which is believed tooccur by ionic binding involving ionic sites on the biopolymer, with thedesired bioactive agent, which may be antimicrobial drugs ormacromolecules such as growth factors, antibacterial agents,antispasmodic agents, or any other active biological bioactive agent,such as adrenergic agents such as ephedrine, desoxyephedrine,phenylephrine, epinephrine and the like, cholinergic agents such asphysostigmine, neostigmine and the like, antispasmodic agents such asatropine, methantheline, papaverine and the like, tranquilizers andmuscle relaxants such as fluphenazine, chlorpromazine, triflupromazine,mephenesin, meprobamate and the like, antidepressants likeamitriptyline, nortriptyline, and the like, antihistamines such asdiphenhydramine, dimenhydrinate, tripelennamine, perphenazine,chlorprophenazine, chlorprophenpyradimine and the like, hyptotensiveagents such as rauwolfia, reserpine and the like, cardioactive agentssuch as bendroflumethiazide, flumethiazide, chlorothiazide, aminotrate,propranolol, nadolol, procainamide and the like, angiotensin convertingenzyme inhibitors such as captopril and enalapril, bronchodialators suchas theophylline, steroids such as testosterone, prednisolone, and thelike, antibacterial agents, e.g., sulfonamides such as sulfadiazine,sulfamerazine, sulfamethazine, sulfisoxazole and the like, antimalarialssuch as chloroquine and the like, antibiotics such as the tetracyclines,nystatin, streptomycin, cephradine and other cephalosporins, penicillin,semi-synthetic penicillins, griseofulvin and the like, sedatives such aschloral hydrate, phenobarbital and other barbiturates, glutethimide,antitubercular agents such as isoniazid and the like, analgesics such asaspirin, acetaminophen, phenylbutazone, propoxyphene, methadone,meperidine and the like, etc. These substances are frequently employedeither as the free compound or in a salt form, e.g., acid additionsalts, basic salts like alkali metal salts, etc. Other therapeuticagents having the same or different physiological activity can also beemployed in the pharmaceutical preparations within the scope of thepresent invention. Typically, the bioactive agent dissolved in asuitable solvent will be contacted with the cross-linked biologicalpolymer by immersion. The loading of the biopolymer may be readilydetermined based upon the uptake of the biopolymer of the bioactiveagent.

[0111] One embodiment of the present invention is to provide a methodfor forming the loaded cross-linked biopolymer, the bioactive agentbeing dissolved in water at a suitable concentration, and thecross-linked biological polymer is immersed therein for an optimizedperiod of time and optimized temperature. The PUH is then extracted fromthe solvent, allowed to air dry or is lyophilized, and is then ready foruse.

[0112] Alternatively, the cross-linked biopolymer may be loaded with thebioactive agent, then dried, then cut to a suitable form for use.

[0113] In another embodiment of the present invention, the bioactiveagent and PUH are dissolved in an aqueous solvent before cross-linkingand the bioactive agent is bound to the biopolymer. The biopolymer isthen cross-linked by treatment with the cross-linking agent.

[0114] It will be realized that the polyubiquitin may be modified, forexample, so as to be made more hydrophilic or hydrophobic to adjust forsuitable binding properties to the bioactive agent. Such modificationmay be performed by, for example, esterification of acid groups in theubiquitin units prior to cross-linking, thus making the ubiquitin morehydrophobic. Another modification relates to recombinant form of thepolyubiquitin, where polypeptide of interest may be placed between tworepeats of ubiquitin unit in the tandem before submitting thecomposition to the cross-linking agents.

[0115] It is an embodiment of the present invention to provide a newdrug delivery system which is easily used which contains a padcomprising a biopolymer which serves as a delivery vehicle forcontrolled release of a bioactive agent to the wound site.

[0116] Another embodiment of the present invention is to serve as adetection device, or for diagnostic purposes. The invention provides astimuli-responsive hydrogel that undergo abrupt changes in volume anddensity in response to external stimuli such, as pH, temperature andsolvent composition that have potential applications in biomedicine andthe creation of intelligent material system, for example as matrix forseparation process and protein process and protein immobilization, or ashybridization-based diagnostic device. Furthermore, the polyubiquitinhydrogel of the invention is responsive to pH, temperature, electricfield, and different other conditions. For some biomedical applications,the polyubiquitin hydrogel is useful by being capable of swelling inresponse dictated by a specific protein.

[0117] When loaded with a detector, that can be an antibody, an antigen,a DNA or RNA fragment, or other molecule that can bind to a biologicalmarker, a targeted molecule to be detected or measured in a biologicalsample, and that may be ubiquitin-linked, the PUH is reported to be ableto swell reversibly in a buffer solution in response to a specificantigen for example. The PUH is previously prepared by grafting theantigen and corresponding antibody to the polymer network, so that thebinding between the two introduces crosslinks in the network.Competitive binding of the free antigen triggers a change in gel volume,density of appearance owing to breaking of these non-covalentcrosslinks.

[0118] The matrix of the present invention may be used as a support forimmunohistochemistry assays.

[0119] One aspect of the present invention is that PUH may display ashape-memory behavior, and that stepwise changes in target moleculeconcentration can induce pulsatile permeation of a protein through thenetwork. The feature is to use the reversible binding between an antigenand an antibody, complementary DNA fragments, or complementary DNA andRNA fragments, as the crosslinking mechanism in thesemi-interpenetrating network hydrogel. The PUH can swell in thepresence of a free targeted molecule, an antigen or nucleotidic fragmentbecause the intra-chain probe-target binding can be dissociated byexchange of the grafted target for free target. In the absence of freetarget, the PUH can shrink. Binding between probes and targets in PUHcan be registered by measurement of optical, density, conductivity, orweight changes.

[0120] In another embodiment of the present invention, thepolynucleotides that may be used include oligonucleotide sequences,complementary RNA and DNA molecules, and PNAs. The polynucleotides maybe used to detect and quantitate gene expression in biopsied tissues.The diagnostic assay may be used to distinguish between absence,presence, and excess expression of biological marker, and to monitorregulation of marker levels during therapeutic intervention.

[0121] According to another embodiment of the present invention,hybridization with PCR probes which are capable of detectingpolynucleotide sequences, including genomic sequences, encoding markersor closely related molecules may be used to identify nucleic acidsequences which encode these markers. The specificity of the probe,whether it is made from a highly specific region (e.g., the 5′regulatory region) or from a less specific region (e.g., the 3′ codingregion), and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding markers, alleles,or related sequences.

[0122] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the marker encoding sequences. The hybridization probes of thesubject invention may be DNA or RNA and may be derived from the sequenceof the marker or from genomic sequences including promoter and enhancerelements and introns of the naturally occurring marker.

[0123] Means for producing specific hybridization probes for DNAsencoding a targeted marker include the cloning of polynucleotidesequences encoding marker or marker derivatives into vectors for theproduction of mRNA probes. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by means of the addition of the appropriate RNA polymerases andthe appropriate labeled nucleotides. Hybridization probes may be labeledby a variety of reporter groups, for example, by radionucleides such asp³² S35, or by enzymatic labels, such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems, and the like.

[0124] Polynucleotide sequences or oligonucleotides may be used in PUHfor the diagnosis of a genetically associated disorder. Disordersinclude, but are not limited to, cancers such as adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; neuronal disorders such as akathesia, Alzheimer's disease,amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia,cerebral neoplasms, dementia, depression, Down's syndrome, tardivedyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, Parkinson's disease, paranoid psychoses, schizophrenia, andTourette's disorder; developmental disorders such as renal tubularacidosis, Cushing's syndrome, achondroplastic dwarfism, Duchenne andBecker muscular dystrophy, gonadal dysgenesis, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spinal bifida, and congenital glaucoma,cataract, or sensorineural hearing loss; and immune disorders such asAddison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,cholecystitis, contact dermayitis, Crohn's disease, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, ulcerative colitis, Werner syndrome,and complications of cancer, hemodialysis, and extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections; and trauma. The polynucleotide sequences encodingmarker may be used in Southern or Northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; in dipstick, pin, andELISA assays; and in microarrays utilizing fluids or tissues frompatient biopsies to detect altered marker expression. Such qualitativeor quantitative methods are known in the art.

[0125] In one embodiment of the invention, the nucleotide sequencesencoding targeted marker may be useful in assays that detect thepresence of associated disorders, particularly those mentioned above.The nucleotide sequences encoding markers may be labeled by standardmethods and added to a fluid or tissue sample from a patient underconditions suitable for the formation of hybridization complexes. Aftera suitable incubation period, the sample is washed and the signal ismeasured and compared with a standard value. If the amount of signal inthe patient sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding markers in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0126] In order to provide a basis for the diagnosis of a disorderassociated with expression of targeted markers, a normal or standardprofile for expression is established. This may be accomplished bycombining body fluids or cell extracts taken from normal subjects,either animal or human, with a sequence, or a fragment thereof, encodingtargeted markers, under conditions suitable for hybridization oramplification. Standard hybridization may be quantified by comparing thevalues obtained from normal subjects with values from an experiment inwhich a known amount of a substantially purified polynucleotide is used.Standard values obtained from normal samples may be compared with valuesobtained from samples from patients who are symptomatic for a disorder.Deviation from standard values is used to establish the presence of adisorder.

[0127] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to evaluate whether the level of expression in the patient beginsto approximate that is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0128] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0129] Additional diagnostic uses for oligonucleotides designed from thesequences encoding markers may involve the use of PCR. These oligomersmay be chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding marker, or a fragment of a polynucleotide complementary to thepolynucleotide-encoding marker, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection ormeasure of closely related DNA or RNA sequences.

[0130] In further embodiments of the present invention, oligonucleotidesor longer fragments derived from any of the polynucleotide sequencesdescribed herein may be used as targets in a microarray. The microarraycan be used to monitor the expression level of large numbers of genessimultaneously (to produce a transcript image) and to identify geneticvariants, mutations, and polymorphisms. This information may be used indetermining gene function, in understanding the genetic basis of adisorder, in diagnosing a disorder, and in developing and monitoring theactivities of therapeutic agents.

[0131] The microarray may be composed of a large number of uniquesingle-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6 to 60 nucleotidesin length, more preferably about 15 to 30 nucleotides in length, andmost preferably about 20 to 25 nucleotides in length. For a certain typeof microarray, it may be preferable to use oligonucleotides that areabout 7 to 10 nucleotides in length. The microarray may containoligonucleotides that cover the known 5′ or 3′ sequence, or may containsequential oligonucleotides which cover the full-length sequence orunique oligonucleotides selected from particular areas along the lengthof the sequence. Polynucleotides used in the microarray may beoligonucleotides specific to a gene or genes of interest in which atleast a fragment of the sequence is known or oligonucleotides specificto one or more unidentified cDNAs common to a particular cell or tissuetype or to a normal, developmental, or disease state. In certainsituations, it may be appropriate to use pairs of oligonucleotides on amicroarray. The pairs will be identical, except for one nucleotidepreferably located in the center of the sequence. The secondoligonucleotide in the pair (mismatched by one) serves as a control. Thenumber of oligonucleotide pairs may range from about 2 to 1,000,000.

[0132] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmthat starts at the 5′ end, or, more preferably, at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In one aspect, the oligomers aresynthesized at designated areas on a substrate using a light-directedchemical process. The substrate may be paper, nylon, any other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

[0133] Fluorescent in situ hybridization (FISH, as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at the Online MendelianInheritance in Man (OMIM) site. Correlation between the location of atargeted gene on a physical chromosomal map and a specific disorder, orpredisposition to a specific disorder, may help define the region of DNAassociated with that disorder. The nucleotide sequences of the subjectinvention may be used to detect differences in gene sequences betweennormal, carrier, and affected individuals.

[0134] In vitro hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc., among normal, carrier, or affected individuals.

[0135] The present invention will be more readily understood byreferring to the following examples that are given to illustrate theinvention rather than to limit its scope.

EXAMPLE I The use of PUH for Realization of Molecular Techniques onSpecimen Deposited on Microscope Slide

[0136] The PUH can be used as a device replacing the humid chamber andthe slide cover in ISH technique (FIG. 1). The PUH is equilibrated witha sodium salt buffer usually citrate buffer (6×SSC). A mixture ofpoly(Adenosine)₁₆ and oligonucleotides specific to the targeted gene areadsorbed to the PUH surface. The specimen, which is either a tissuesection, individual cells or nucleic acid, is mounted on a support suchas microscope slide. The PUH mounted on a plastic support is thenapplied on the specimen. The microscope slide is incubated at 95° C. for2 min and cooled to the hybridization temperature. The incubation timefor hybridization is determined empirically and is sufficient to allowthe oligonucleotides to hybridize with the target gene. Afterhybridization the PUH is peeled off from the microscope slide andreplaced by a new PUH previously equilibrated with a stringent saltbuffer and incubated for 10 min. This wash step removes non-specificinteractions of the probe. The PUH is removed and the slides areprocessed for probe detection. The procedures for probe detection varyupon the label used (e.g. radioactive, fluorescence, biotin,digoxigenin).

EXAMPLE II Preparation of Biosensors with PUH

[0137] The monoubiquitin (1 unit) or polyubiquitin (2 to 6 units) wassuspended in different pH buffers: PBS (potassium phosphate 100 mM, 150mM NaCl, pH 7,4), Borate buffer (boric acid 50 mM, 100 mM NaCl, pH 8,0)or Carbonate buffer (sodium bicarbonate 100 mM, pH 9,4) at concentrationranging between 1 to 100 mg/ml. A polyethylene bis-p-nitrophenylcarbonate (PEG) solution ranging between 10 to 100 mg/ml suspended inrespective above buffers was mixed in 1:1 proportion to the ubiquitinsolution and incubated 2 to 16 hours at room temperature. Highconcentration of mono or polyubiquitin (>5% w/v) hydrogels polymerizedin carbonate buffer gave solid transparent polymers as shown in FIG. 2.FIG. 3 illustrates the molecular network relation between units ofubiquitin during gel formation. To perform ultrastructural analysisafter polymerization, the PUH were fixed in 4% v/v formaldehyde incacodylate buffer (100 mM, pH 7,3). They were rinsed three times withthe cacodylate buffer and post-fixed with osmium tetroxide 1% in thesame buffer for 90 min at room temperature. The PUH were then dehydratedin alcohol and embedded in LRWhite resin (Marivac, Halifax, Canada).Ultra thin sections were deposited on formvar coated nickel grids,stained uranyl acetate and lead citrate. Sections were assessed andphotographed using a Joel 1200-EX electron microscope at a voltage of 80kV. Ultrastructure of a PUH (2% polyubiquitin hexamer, 10% PEG M.W.8000) is represented in FIG. 4.

[0138] Low concentration of polyubiquitin (<2% w/v) hydrogelspolymerized in Borate buffer gave hydrogel spheres. To determine thesize of spheres, these hydrogels were fixed and dehydrated as describedabove and a drop was air dried on an aluminum SEM stub using doublesided carbon adhesive disks. The stub was then gold coated in a sputtercoating unit for 10 min with 20 nm of gold. Spheres were examined andphotographed with a JSM 35CF field emission scanning electron microscopeat accelerating voltages of 15-20 kV. The PUH sphere diameters (2%polyubiquitin hexamers, 10% PEG M.W. 8000) were less then 1 μM as shownin FIG. 5. FIG. 6 shows a macroscopic view of the network that can beformed in polymerizing units of ubiquitin with antibodies.

EXAMPLE III Optical Properties of PUH

[0139] Hexamer and monomer of ubiquitin suspended in PBS pH 7.4 weresubmitted to a optical density scan (absorbency) ranging between 220 to600 nm with 1 nm stepwise. Bovine albumin serum (BSA) was used as acontrol. Polyubiquitin showed a distinctive absorbency pattern in UVspectrum whereas monomer of ubiquitin has a similar absorbency profileof BSA with a typical absorbency peak near 280 nm (FIG. 7). Also, PUHshow a constant transmittance of light at different wavelength, whileBSA gels gives variable transmittance of light (FIG. 8). The PUH wasthen introduced in a quartz cell in presence of PBS pH 7.4. Thespectrophotometer cell holder temperature was controlled by acirculating bath. Variation of temperature from 20° C. to 60° C. wasperformed by 10° C. stepwise. The biopolymer was stabilized 5 minutes ateach temperature steps before full spectrum scan was performed. The PUHabsorbency profile was similar to the polyubiquitin in solution. Theoptical density (absorbency) in UV spectrum varied upon temperaturechanges as shown in FIG. 9. A plot of the absorbency against temperatureshowed a direct linear relation between 30° C. and 60° C. (FIG. 10). Theresponse of PUH to salt was performed by adding 200 μl of 5M NaCl. Timecourse readings were taken at 10 minutes intervals for 1 hour. Theoptical density (absorbency) in UV spectrum varied upon salt changes asshown in FIG. 11. The optical density changed rapidly and a plateau wasobserved after 30 min as shown in FIG. 12.

[0140] Ubiquitin based hydrogel polymerized (5% w/v polyubiquitin 6units, 12% w/v PEG 8000 M.W.) in 96 well plates were washed andequilibrated with three different buffers: Na-Citrate (100 mM sodiumcitrate, 150 mM NaCl, pH 5.2), PBS (100 mM potassium phosphate, 150 mMNaCl, pH 7.4) and Carbonate (100 mM sodium bicarbonate, 150 mM NaCl, pH9.4). Bovine Serum Albumin based (BSA) hydrogel were also polymerized inthe same manner and used as a comparative control. Variousconcentrations of a R-phycoerythrin (PE) conjugated normal goat IgG(Caltag) diluted in the above buffers were placed onto the hydrogels andincubated for one hour at 4° C. The hydrogels were then washed andIgG-PE binding was measured on a fluoroskan Ascent fluorometer(Labsystems OY, Helsinki, Finland) between each wash. The PUH showed astrong binding activity to IgG-PE compared to BSA based hydrogel at highpH and lesser binding activity was observed at neutral and low pH (FIG.13). The IgG-PE bound to the hydrogels were washed out by adding anexcess of unlabelled IgG.

EXAMPLE IV Immobilization of Peroxydase in PUH Nanospheres

[0141] The polyubiquitin (hexamer) and monoubiquitin (monomer) wassuspended in Borate buffer (boric acid 50 mM, 100 mM NaCl, pH 8.0) (100mg/ml and 10 mg/ml respectively). The Horseradish peroxydase (HRP) wassuspended at 20 mg/ml in borate buffer. A polyethylene bis-p-nitrophenylcarbonate (PEG) solution at 10 mg/ml in Borate buffer was mixed in 1:1proportion to the HRP solution and incubated for 10 min at roomtemperature. The polyubiquitin solution was then mixed at equal 1:1:1ratio with the PEG:HRP solution and incubated at 22° C. for 16 h. Thenanospheres suspension was then centrifuged at 14 000 g in amicrocentrifuge for 10 min and suspended and washed three times inPhosphate Buffered Saline (PBS) pH 7.4. The HRP immobilization wasrevealed by adding the AEC substrate (Signet Laboratories Inc.), 0.3%v/v H202 to the nanospheres. After 10-min incubation, centrifuging themicrospheres and suspending them in PBS stopped the developing solution.The stained nanospheres where then observed under microscope at 600×(FIG. 14). The relation between ligand molecule (a), immobilized enzymes(b) and PUH is illustrated in FIG. 15. HRP activity was measured byadding o-phenylenediamine dihydrochloride (OPD) in PBS, 0.3% v/v H₂O₂ toa serial dilution of PUH nanospheres suspension in a 96 wells plate. Theplate was then read in a Thermomax™ microplate reader using theSOFTmaxPro™ software (Molecular Devices, Sunnyvale, Calif.). At leastthree counts of absorbency per wells were performed at 550 nm. Allvalues have been corrected for the optical density of the substratesolution (OPD) (FIG. 16).

EXAMPLE V PUH as In vivo Delivery System

[0142] The polyubiquitin (hexamer) was suspended in a carbonate bufferat 100 mg/ml. Insulin labeled with 25 μCi 125-iodine (specific activityof 50 mCi/ml) was mixed with the polyubiquitin (6 units) solution.Polyethylene bis-p-nitrophenyl carbonate (PEG M.W. 8000) solution at 250mg/ml in carbonate buffer pH 9.4 was then mixed with thepolyubiquitin-insulin solution and incubated for 16 h at 22° C. ThePUH-125I-insulin was then washed extensively in PBS solution to removeall traces of phenol. The PUH-125I-insulin conjugate was crushed into a18 gauge needle using a 3 cc syringe.

[0143] Six male adult Sprague-Dawley rats weighing approximately 200 gwere divided in two groups of three rats. The control group of ratsreceived 5 μCi of free 125I-labeled insulin each. The test group of ratsreceived the same amount of 125I-labeled insulin immobilized in the PUH.Four days prior to and during the experiment, rats drank an aqueoussolution of potassium iodine (20 mM). After intradermic administrationof 125I-labeled insulin free or immobilized, blood samples were takenout at time 0 h, 2 h, 4 h, 6 h, 24 h 48 h. gamma-(125I) radioactivitieswere counted in a gamma-scintillation counter. Results were expressed aspercentages of the administered amount of radioactivity per ml of bloodsampled. FIG. 17 shows a delayed release of PUH immobilized insulin inthe venous blood compared to free insulin.

EXAMPLE VI The use of PUH for Realization of Molecular Techniques onSpecimen Deposited on Microscope Slide

[0144] Immunohistochemistry

[0145] Normal kidney specimens were fixed with formalin and embedded inparaffin. Sections of 5 μm were placed on charged glass slides(Surgipath™, Winnipeg, Manitoba), deparaffinized and rehydrated usingxylene, graded ethanol and PBS. Background sample peroxidase activitywas inhibited with a 3% H₂O₂ solution for 5 min (Signet Laboratories,Dedham, Mass.). Non-specific IgG interaction were reduced by incubatingsections with normal serum for 5 min (Signet Labs). Sections werestained for 60 minutes with 20 μl (1:500 dilution) of mouse anti-humanEpithelial Membrane Antigen (EMA, clone E29, Signet Labs.). The slideswere covered either with a micro cover glass and placed in a humidifyingchamber or with the PUH prototype (FIGS. 1 and 18). The slides in thehumidity chamber were washed with PBS prior to detection while the onescovered with the PUH were directly used for detection after removal ofthe PUH prototype. Staining was revealed using the Level 2 multi-speciesUltra Streptavidin HRP Detection System and AEC (Signet Labs). Slideswere counterstained with Harris modified hematoxylin (Fisher) andmounted with ultramount (DAKO Diagnostics Canada). FIG. 19 shows aspecific staining of the epithelial cells.

EXAMPLE VI

[0146] PUH as control release system of steroids The polyubiquitinhydrogel in PBS pH 7,4 was equilibrated in a dexamethasone solution at 4mg/ml. After 2 h incubation, the PUH-dexamethasone was washed with PBSand used immediately or dehydrated at 37° C. for 16 h. A peristalticpump with a flow rate of 25 cc/min was used to circulate a PBS solutionfrom a diffusion chamber connected to a flow cell unit. Thespectrophotometer cell holder temperature was controlled by acirculating bath. The absorbency was measured at 255 nm continuously upto 90 min. After 1 min of readings, the PUH-dexamethasone was added inthe diffusion chamber. The FIG. 20 shows a rapid release ofdexamethasone with the hydrated PUH and a delayed release with thedehydrated PUH.

[0147] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1 1 1 76 PRT unknown Ubiquitin 1 Met Gln Ile Phe Val Lys Thr Leu Thr GlyLys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu AsnVal Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln GlnArg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu SerAsp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg LeuArg Gly Gly 65 70 75 1/1

What is claimed is:
 1. A biopolymer comprising a mixture of ubiquitinand at least one cross-linking agent.
 2. A biopolymer according to claim1, wherein said cross-linking agent is selected from the groupconsisting of a photoreactive cross-linking agent and a thermoreactivecross-linking agent.
 3. A biopolymer according to claim 2, wherein saidthermoreactive cross-linking agent is a compound containing athermochemical reactive group selected from the group consisting of:—COOH (carboxylic acids), sulfonic acid derivatives, —COOR (esters),—COX (acid halides, acid azides and similar carboxylic acidderivatives), —CONHNH₂ (acid hydrazides), —NHCONHNH₂ (semicarbazides),—NHCSNHNH₂ (thiosemicarbazides), —CHO (aldehydes), RR′CO (ketones), —OH(alcohols), —X (halides: chloride, bromide, iodide), —SH thiols, —SSR(disulfides), —NH₂ (primary amines), —NH— (secondary amines), —N—(tertiary amines), —NHNH₂ (hydrazines), epoxides, and maleimides.
 4. Abiopolymer according to claim 1, wherein said ubiquitin comprises atleast one ubiquitin unit.
 5. A biopolymer according to claim 1, whereinsaid mixture comprises ubiquitin units in tandem.
 6. A biopolymeraccording to claim 5, wherein said mixture comprises between 2 to about25 ubiquitin units and combination thereof.
 7. A biopolymer according toclaim 4, wherein said tandem comprises 7 ubiquitin units.
 8. Abiopolymer according to claim 1, wherein said mixture comprises at leastone ubiquitin selected from the group consisting of recombinantubiquitin, naturally occurring ubiquitin, mutant, analog, fragment, andderivative thereof.
 9. A biopolymer according to claim 1, wherein saidcross-linking agent comprises a polyethylene glycol, a derivative ofpolyethylene glycol, or a mixture thereof.
 10. A biopolymer according toclaim 1, wherein said cross-linking agent is selected from the groupconsisting of polyamine, amine, polyvinyl, polystyrene, epoxy, silicone,proteinaceaous, keratin, collagen, elastin, actin, myosin, fibrinogen,silk, polysaccharides, cellulose, amylose, hysluronic acid, gelatin,chitin, chitosan, xylan, mannan, silica, p-Azidobenzoyl hydrazide,N-5-Azido-2-nitrobenzoyloxysuccinimide, disuccinimidyl glutamate,dimethyl pimelimidate-2 HCL, dimethyl suberimidate-2 HCL,dithiiobis[succiniiimidyl propionate], disuccinimidyl suberate,bis[sulfosuccinimidyl suberate], 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide HCL, isocyanate, aldhyde, glutaraldehyde, paraformaldehyde,and derivative thereof.
 11. A biopolymer according to claim 9, whereinsaid cross-linking agent comprises a derivative of polyethylene glycol.12. A biopolymer according to claim 9, wherein said derivative isselected from the group consisting of polyethylene oxide of the generalformula 1: X—(CH₂—CH₂—O)_(n)—X wherein n is at least 1; X is a covalentbound or capable of reacting with an amino acid, or is an R or ROradical in which the oxygen is bound to the polyethylene oxide and R isselected from the group selected from the group of methylene, ethylene,propylene, o-, m- and p-phenylene, o-, m- and p-phenylene carbamateunsubstituted or substituted by at least one alkyl, aryl, halo, nitro,oxo, carboxy, hydroxy, thio, sulfonate, hydroxy and phosphate groups.13. A biopolymer according to claim 9, wherein said derivative comprisesan activated bifunctionalized polyethylene oxide.
 14. A process forpreparing a ubiquitin biopolymer comprising the steps of: a) mixing aubiquitin solution with at least one cross-linking agent, and b)inducing polymerization between said ubiquitin in solution and saidcross-linking agent of step a) for a time sufficient for a cross-linkingreaction to occur.
 15. A process according to claim 14, wherein saidbiopolymer comprises a ubiquitin unit.
 16. A process according to claim14, wherein said cross-linking agent is selected from the groupconsisting of photoreactive linking agent and thermoreactivecross-linking agent.
 17. A process according to claim 16, wherein saidthermoreactive linking agent is a compound containing a thermochemicalreactive group selected from the group consisting of: —COOH (carboxylicacids), sulfonic acid derivatives, —COOR (esters), —COX (acid halides,acid azides and similar carboxylic acid derivatives), —CONHNH₂ (acidhydrazides), —NHCONHNH₂ (semicarbazides), —NHCSNHNH₂(thiosemicarbazides), —CHO (aldehydes), RR′CO (ketones), —OH (alcohols),—X (halides: chloride, bromide, iodide), —SH thiols, —SSR (disulfides),—NH₂ (primary amines), —NH— (secondary amines), —N— (tertiary amines),—NHNH₂ (hydrazines), epoxides, and maleimides.
 18. A process accordingto claim 14, wherein said biopolymer comprises a tandem of ubiquitinunits.
 19. A process according to claim 14, wherein said biopolymercomprises tandem composed of between about 2 to 25 ubiquitin units andcombination thereof.
 20. A process according to claim 19, wherein saidbiopolymer comprises tandem composed of 7 ubiquitin units.
 21. A processaccording to claim 14, wherein said biopolymer comprises at least oneubiquitin selected from the group consisting of recombinant ubiquitin,naturally occurring ubiquitin, mutant, analog, fragment, and derivativethereof.
 22. A process according to claim 14, wherein said cross-linkingagent comprises a polyethylene glycol.
 23. A process according to claim22, wherein said cross-linking agent comprises polyethylene glycol,derivative of polyethylene glycol, or a mixture thereof.
 24. A processaccording to claim 23, wherein said derivative is selected from thegroup consisting of polyethylene oxide of the general formula 1:X—(CH₂—CH₂—O)_(n)—X wherein n is at least 1; X is a covalent bound orcapable of reacting with an amino acid, or is an R or RO radical inwhich the oxygen is bound to the polyethylene oxide and R is selectedfrom the group selected from the group of methylene, ethylene,propylene, o-, m- and p-phenylene, o-, m- and p-phenylene carbamateunsubstituted or substituted by at least one alkyl, aryl, halo, nitro,oxo, carboxy, hydroxy, thio, sulfonate, hydroxy and phosphate groups.25. A process according to claim 24, wherein said derivative comprisesan activated bifunctionalized polyethylene oxide.
 26. A processaccording to claim 14, wherein said cross-linking agent is selected fromthe group consisting of polyamine, amine, polyvinyl, polystyrene, epoxy,silicone, proteinaceaous, keratin, collagen, elastin, actin, myosin,fibrinogen, silk, polysaccharides, cellulose, amylose, hysluronic acid,gelatin, chitin, chitosan, xylan, mannan, silica, p-Azidobenzoylhydrazide, N-5-Azido-2-nitrobenzoyloxysuccinimide, disuccinimidylglutamate, dimethyl pimelimidate-2 HCL, dimethyl suberimidate-2 HCL,dithiiobis[succiniiimidyl propionate], disuccinimidyl suberate,bis[sulfosuccinimidyl suberate], 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide HCL, isocyanate, aldhyde, glutaraldehyde, paraformaldehyde,and a derivative thereof.
 27. A biopolymer consisting essentially ofubiquitin, acceptable solvent of ubiquitin and at least onecross-linking agent.
 28. A biopolymer according to claim 26, whereinsaid biopolymer comprises a ubiquitin unit.
 29. A biopolymer accordingto claim 26, wherein said biopolymer comprises ubiquitin units intandem.
 30. A biopolymer according to claim 26, wherein said biopolymercomprises tandem composed of between about 2 to 25 ubiquitin units andcombination thereof.
 31. A biopolymer according to claim 26, whereinsaid biopolymer comprises tandem composed of 7 ubiquitin units.
 32. Abiopolymer according to claim 26, wherein said ubiquitin comprises atleast one ubiquitin selected from the group consisting of recombinant,mutant, analog, fragment, and a derivative thereof.
 33. Use of ubiquitinin the preparation of a biopolymer as described in claim
 1. 34. The useaccording to claim 33, wherein said biopolymer comprises at least oneubiquitin unit.
 35. The use according to claim 33, wherein saidbiopolymer comprises ubiquitin units in tandem.
 36. The use according toclaim 33, wherein said biopolymer comprises tandem composed of betweenabout 2 to 25 ubiquitin units and combination thereof.
 37. The useaccording to claim 36, wherein said biopolymer comprises tandem composedof 7 ubiquitin units.
 38. The use according to claim 33, wherein saidbiopolymer comprises at least one ubiquitin selected from the groupconsisting of recombinant ubiquitin, naturally occurring ubiquitin,mutant, analog, fragment, and a derivative thereof.